Identification and characterization of aldehyde dehydrogenases (ALDHs) and their suitability as drug targets against the liver fluke Fasciola hepatica

Abstract

The liver fluke Fasciola hepatica is a threat for both animal and human health. Fasciolosis causes economic losses in cattle and sheep farming worldwide, and it has been listed as a neglected tropical disease of humans with >2.4 million people being infected worldwide. Several reports showed the spread of resistance against the main effective drug, triclabendazole. Therefore, finding alternative treatment options is highly demanded. Aldehyde dehydrogenases (ALDHs) are involved in the cellular detoxification of reactive aldehydes. Our real-time qPCR analyses, using two different sets of newly established reference genes, have shown that ALDH orthologues (Fhaldh1 and Fhaldh2) are expressed by various life stages of F. hepatica. We detected Fhaldh1 transcripts in gonadal and gastrodermis tissues using in situ hybridization. Therefore, we next assessed the possible fasciolicidal effects of the known ALDH inhibitor disulfiram, an approved drug for use in humans. Newly excysted juveniles (NEJs), immature, and adult flukes were exposed for three days to disulfiram. Disulfiram in a concentration of 20 µM led to severe effects on fluke motility and tegument integrity. Overall efficiency was even enhanced when a novel disulfiram derivative, with lower cytotoxicity in vitro, was used, which showed severe effects on NEJs already at 2 µM. This derivative (028) exhibited improved efficacy also against immature and adult flukes, which was comparable to that of triclabendazole in our in vitro-culture conditions. Both compounds triggered the expression of oxidative stress-related genes. However, only 028 showed the capacity to abolish cell proliferation in juvenile flukes, which points to an impairment of cell-cycle control. A preliminary result of a double knockdown of both orthologues pointed to a possible role of ALDHs in controlling the growth of juvenile F. hepatica. Altogether, this work identified and characterized to some extent two ALDH orthologues of F. hepatica by in silico analyses as well as molecular and biochemical approaches. Furthermore, this work provides the first evidence that targeting ALDHs could be considered a novel strategy in the fight against F. hepatica. These results provide the basis to continue investigating the role(s) of ALDHs in the biology of F. hepatica in more detail in future studies and developing ALDH inhibitors as anthelmintic drugs.